WO2020028443A1 - Variants d'alpha-amylases ayant des substitutions d'acides aminés qui abaissent le pka de l'acide général - Google Patents

Variants d'alpha-amylases ayant des substitutions d'acides aminés qui abaissent le pka de l'acide général Download PDF

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WO2020028443A1
WO2020028443A1 PCT/US2019/044254 US2019044254W WO2020028443A1 WO 2020028443 A1 WO2020028443 A1 WO 2020028443A1 US 2019044254 W US2019044254 W US 2019044254W WO 2020028443 A1 WO2020028443 A1 WO 2020028443A1
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Prior art keywords
amylase
amino acid
variant
acid sequence
seq
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Jonathan K. LASSILA
Patrica TRAN
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Danisco US Inc
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Danisco US Inc
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Priority to US17/264,531 priority Critical patent/US12584119B2/en
Priority to JP2021505722A priority patent/JP7530884B2/ja
Priority to MX2021001213A priority patent/MX2021001213A/es
Priority to CA3108284A priority patent/CA3108284A1/fr
Priority to EP19758538.3A priority patent/EP3830231A1/fr
Priority to CN201980064393.3A priority patent/CN112805361A/zh
Publication of WO2020028443A1 publication Critical patent/WO2020028443A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • compositions and methods relating to variant a-amylases are useful, for example, for starch liquefaction and saccharification, cleaning starchy stains, textile desizing, baking and brewing.
  • Starch consists of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w).
  • Amylose consists of linear chains of a-l,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000.
  • MW molecular weight
  • Amylopectin is a branched polymer containing a- 1,6 branch points every 24-30 glucose units; its MW may be as high as 100 million.
  • a-amylases hydrolyze starch, glycogen, and related polysaccharides by cleaving internal a-l,4-glucosidic bonds at random a-amylases, particularly from Bacilli, have been used for a variety of different purposes, including starch liquefaction and saccharification, textile desizing, starch modification in the paper and pulp industry, brewing, baking, production of syrups for the food industry, production of feed-stocks for fermentation processes, and in animal feed to increase digestability. These enzymes can also be used to remove starchy soils and stains during dishwashing and laundry washing.
  • compositions and methods relate to variant a-amylase polypeptides, and methods of use, thereof, and methods for designing variant a-amylases. Aspects and embodiments of the present compositions and methods are summarized in the following separately-numbered paragraphs:
  • a recombinant variant of a parental Family 13 a-amylase having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 and having an amino acid mutation in an amino acid residue that lines the N-terminal side of the core b-barrel structure of the a-amylase that results in the variant having an amino acid residue that is different from the naturally occurring amino acid in the parent a-amylase, and which results in reduction of the apparent p a value of the general acid and an increase in the activity of the variant at a pH between about 8.5 and 10.5.
  • the ratio of the activity at pH 8.5 compared to the activity at pH 10.5 for the variant divided by the ratio of the activity at pH 8.5 compared to the activity at pH 10.5 of an otherwise identical a-amylase lacking the amino acid mutation in an amino acid residue that lines the N-terminal side of the core b-barrel structure of the a-amylase is greater than 1, greater than 2, greater than 3, or greater than 4.
  • the variant a-amylase of paragraph 1 or 2 comprises an amino acid substitution at a position selected from the group consisting of T40, F263, S288 and Y364 corresponding to the amino acid sequence of SEQ ID NO: 1.
  • the variant comprises an amino acid substitution selected from the group consisting of T40C, T40D, T40E, T40N, F263P, F263Y, S288D, S288K, Y364E, Y364L and Y364M corresponding to the amino acid sequence of SEQ ID NO: 1.
  • the variant a-amylase of any of paragraphs 1-4 further comprises:
  • the variant a-amylase of any of paragraphs 1-5 has at least 70%, at least 80%, at least 90% or at least 95% amino acid sequence identity to the amino acid sequence of of SEQ ID NO: 1; encoded by a polynucleotide having at least 60%, at least 70%, at least 80% or at least 90% nucleic acid sequence identity to a polynucleotide encoding SEQ ID NO: 1; and/or encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding SEQ ID NO: 1, or the complement, thereof.
  • a method for modulating the acitivity of an a-amylase comprising introducing into a parental Family 13 a-amylase having at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 a mutation of a residue that affects the electrostatic environment of the general acid positioned for catalysis, wherein the mutation is located at the N-terminal side of the a-amylase B-barrel, and wherein the mutation changes the electrostatic environment of the general acid in the resulting variant a-amylase.
  • the ratio of the activity of the variant a-amylase at pH 8.5 compared to the activity at pH 10.5 divided by the ratio of the activity at pH 8.5 compared to the activity at pH 10.5 of an otherwise identical a-amylase lacking the amino acid mutation in an amino acid residue that lines the N-terminal side of the core b-barrel structure of the a-amylase is greater than 1, greater than 2, greater than 3, or greater than 4.
  • the mutation is a substitution at a position selected from the group consisting of T40, F263, S288 and Y364 corresponding to the amino acid sequence of SEQ ID NO: 1.
  • the substitution is selected from the group consisting of T40C, T40D, T40E, T40N, F263P, F263Y, S288D, S288K, Y364E, Y364L and Y364M corresponding to the amino acid sequence of SEQ ID NO: 1.
  • the variant a- amylase further comprises:
  • composition for liquefying starch comprising the variant a- amylase of any of paragraphs 1-6 is provided.
  • a detergent composition comprising the variant amylase of any of paragraphs 1-6 is provided. 14.
  • a method for converting starch to oligosaccharides comprising contacting starch with effective amount of the variant amylase of any of paragraphs 1 6
  • a method for removing a starchy stain or soil from a surface comprising contacting the surface with an effective amount of the variant amylase of any of paragraphs 1-6, and allowing the polypeptide to hydrolyze starch components present in the starchy stain to produce smaller starch-derived molecules that dissolve in the aqueous composition, thereby removing the starchy stain from the surface.
  • Figure 1 is an amino acid sequence alignment of BspAmy24, Amy707 and AA560.
  • Figure 2 shows a structural model of BspAmy24 (del-Rl8l-Gl82) for viewing with conventional stereo viewer.
  • Figure 3 shows the structural model of BspAmy24 (del-Rl8l-Gl82) from a different angle.
  • the a carbon positions are shown in black for residues 40, 41, 263, 288, and 364.
  • the general acid residues are shown as gray spheres.
  • Figure 4 is a plot of the log of rate constant versus pH for BspAmy24-Vl and
  • Figure 5 is a plot of the log of rate constant versus pH for BspAmy24-Vl and
  • Figure 6 is a plot of the log of rate constant versus pH for BspAmy24-Vl and
  • Figure 7 is a plot of the log of rate constant versus pH for BspAmy24-Vl and
  • compositions and methods relating to variant a-amylase enzymes are described.
  • Exemplary applications for the variant amylase enzymes are for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing, and other applications, for textile processing ( e.g ., desizing), in animal feed for improving digestibility, and and for baking and brewing.
  • amylase or“amylolytic enzyme” refer to an enzyme that is, among other things, capable of catalyzing the degradation of starch a-amylases are hydrolases that cleave the a-D-(l 4) O-glycosidic linkages in starch.
  • a-amylases (EC 3.2.1.1; a-D-(l 4)- glucan glucanohydrolase) are defined as endo-acting enzymes cleaving a-D-(l 4) O-glycosidic linkages within the starch molecule in a random fashion yielding polysaccharides containing three or more (l-4)-a-linked D-glucose units.
  • the exo-acting amylolytic enzymes such as b-amylases (EC 3.2.1.2; a-D-(l 4)-glucan maltohydrolase) and some product-specific amylases like maltogenic a-amylase (EC 3.2.1.133) cleave the polysaccharide molecule from the non-reducing end of the substrate b-amylases, a-glucosidases (EC 3.2.1.20; a-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; a-D-(l 4)-glucan glucohydrolase), and product- specific amylases like the maltotetraosidases (EC 3.2.1.60) and the maltohexaosidases (EC 3.2.1.98) can produce malto-oligosaccharides of a specific length or enriched syrups of specific maltooligosaccharides.
  • starch refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (GHioOsjx, wherein X can be any number.
  • the term includes plant-based materials such as grains, cereal, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, milo, potato, sweet potato, and tapioca.
  • the term“starch” includes granular starch.
  • the term“granular starch” refers to raw, i.e., uncooked starch, e.g. , starch that has not been subject to gelatinization.
  • the term“liquefaction” or“liquefy” means a process by which starch is converted to less viscous and shorter chain dextrins.
  • the terms,“wild-type,”“parental,” or“reference,” with respect to a polypeptide refer to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions.
  • the terms“wild-type,”“parental,” or “reference,” with respect to a polynucleotide refer to a naturally-occurring polynucleotide that does not include a man-made nucleoside change.
  • a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring
  • polynucleotide encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.
  • A“mature” polypeptide or variant, thereof, is one in which a signal sequence is absent, for example, cleaved from an immature form of the polypeptide during or following expression of the polypeptide.
  • polypeptide refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally-occurring or man-made substitutions, insertions, or deletions of an amino acid.
  • the term“variant,” with respect to a polynucleotide refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.
  • “activity” refers to a-amylase activity, which can be measured as described herein.
  • core b-barrel structure refers to the amino acid residues that form a central region of parallel b-sheet secondary structure, curving upon itself such that the first strand hydrogen bonds with the last strand in a continuous barrel.
  • amino acids that form the barrel are residues 8-11, 40-44, 98-103, 232-235, 263-267, 288- 290, 325-328, 364-368.
  • an amino acid residue that lines the N-terminal side of the core b-barrel structure refers to an amino acid at the end of the core b-barrel structure that is opposite to the side that is adjacent to the active site.
  • the strands that comprise the barrel are oriented in parallel, such that the N-terminal ends of the b strands lie on one side of the barrel and the C- terminal ends of the strands lie on the other side of the barrel, adjacent to the active site.
  • the amino acids that line the N-terminal side of the core b -barrel include the most N-terminal residues of each strand that are engaged in hydrogen bonding with adjacent strands, as defined by common algorithms to assign secondary structure such as in PyMol and MOE, as well as the residue immediately before or after those residues if engaged in backbone hydrogen bonding with an adjacent strand.
  • the residues that line the N-terminal side of the core b-barrel structure in BspAmy24 include positions 8, 9, 40, 41, 97, 98, 230, 232, 263, 288, 325, 326, 364.
  • the apparent p K a of the general acid refers to the experimentally determined value for the higher p K a of the enzyme’s activity, such that at pH values above the p/fa, the activity decreases with increasing pH, with a roughly one order of magnitude drop in activity for each pH unit at values well above the p K a .
  • This observed p/fa is expected to correspond to that of the general acid in the reaction mechanism (Rydberg, E.H. et al. (2002) Mechanistic analyses of catalysis in human pancreatic a-amylases: Detailed kinetic and structural studies of three conserved carboxylic acids. Biochemistry 41 :4492-4502), yet it is also acknowldeged that kinetic properties of the enzyme and other ionizations may complicate the direct assignment.
  • performance benefit refers to an improvement in a desirable property of a molecule.
  • exemplary performance benefits include, but are not limited to, increased hydrolysis of a starch substrate, increased grain, cereal or other starch substrate liquifaction performance, increased cleaning performance, increased thermal stability, increased detergent stability, increased storage stability, increased solubility, an altered pH profile, decreased calcium dependence, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, and increased expression.
  • the performance benefit is realized at a relatively low temperature.
  • the performance benefit is realized at relatively high temperature.
  • variants are variants comprising two or more mutations, e.g ., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, substitutions, deletions, and/or insertions.
  • recombinant when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified from its native state.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
  • Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g. , a heterologous promoter in an expression vector.
  • Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences.
  • a vector comprising a nucleic acid encoding an amylase is a recombinant vector.
  • polypeptides include, but is not limited to, a culture broth containing secreted polypeptide expressed in a heterologous host cell.
  • purified refers to material (e.g, an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g, at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.
  • thermostability refers to the ability of the enzyme to retain activity after exposure to an elevated temperature.
  • the thermostability of an enzyme such as an amylase enzyme, is measured by its half-life (ti/2) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring residual a-amylase activity following exposure to (i.e., challenge by) an elevated temperature.
  • A“pH range,” with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.
  • the terms“pH stable” and“pH stability,” with reference to an enzyme, relate to the ability of the enzyme to retain activity over a wide range of pH values for a predetermined period of time (e.g ., 15 min., 30 min., 1 hour).
  • amino acid sequence is synonymous with the terms“polypeptide,”“protein,” and“peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an“enzyme.”
  • amino acid sequences exhibit activity, they may be referred to as an“enzyme.”
  • the conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino- to-carboxy terminal orientation (i.e., N C).
  • nucleic acid encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may contain chemical modifications.
  • nucleic acid and
  • polynucleotide are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.
  • Hybridization refers to the process by which one strand of nucleic acid forms a duplex with, i.e., base pairs with, a complementary strand, as occurs during blot hybridization techniques and PCR techniques.
  • Hybridized, duplex nucleic acids are characterized by a melting temperature (Tm), where one half of the hybridized nucleic acids are unpaired with the complementary strand.
  • A“synthetic” molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism.
  • the terms“transformed,”“stably transformed,” and“transgenic,” used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.
  • a non-native nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.
  • the term“introduced” in the context of inserting a nucleic acid sequence into a cell means“transfection”,“transformation” or“transduction,” as known in the art.
  • A“host strain” or“host cell” is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g ., an amylase) has been introduced.
  • Exemplary host strains are microorganism cells (e.g, bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides.
  • the term“host cell” includes protoplasts created from cells.
  • heterologous with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.
  • endogenous with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.
  • the term“expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.
  • specific activity refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U)/mg of protein.
  • water hardness is a measure of the minerals (e.g, calcium and magnesium) present in water.
  • A“swatch” is a piece of material such as a fabric that has a stain applied thereto.
  • the material can be, for example, fabrics made of cotton, polyester or mixtures of natural and synthetic fibers.
  • the swatch can further be paper, such as filter paper or nitrocellulose, or a piece of a hard material such as ceramic, metal, or glass.
  • the stain is starch based, but can include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigment, oil, or mixtures of these compounds.
  • A“smaller swatch” or“micro swatch” is a section of the swatch that has been cut with a single hole punch device, or has been cut with a custom manufactured multiple-hole punch device, where the pattern of the multi-hole punch is matched to standard multi-well microtiter plates, or the section has been otherwise removed from the swatch.
  • the swatch can be of textile, paper, metal, or other suitable material.
  • the smaller swatch can have the stain affixed either before or after it is placed into the well of a 24-, 48- or 96-well microtiter plate.
  • the smaller swatch can also be made by applying a stain to a small piece of material.
  • the smaller swatch can be a stained piece of fabric 5/8" or 0.25" or 5.5 mm in diameter.
  • the custom manufactured punch is designed in such a manner that it delivers 96 swatches simultaneously to all wells of a 96-well plate.
  • the device allows delivery of more than one swatch per well by simply loading the same 96-well plate multiple times.
  • Multi-hole punch devices can be conceived of to deliver simultaneously swatches to any format plate, including but not limited to 24-well, 48-well, and 96-well plates.
  • the soiled test platform can be a bead made of metal, plastic, glass, ceramic, or another suitable material that is coated with the soil substrate.
  • the one or more coated beads are then placed into wells of 96-, 48-, or 24-well plates or larger formats, containing suitable buffer and enzyme.
  • the stained fabric is exposed to enzyme by spotting enzyme solution onto the fabric, by wetting swatch attached to a holding device, or by immersing the swatch into a larger solution containing enzyme.
  • a cultured cell material comprising an amylase refers to a cell lysate or supernatant (including media) that includes an amylase as a component.
  • the cell material may be from a heterologous host that is grown in culture for the purpose of producing the amylase.
  • “Percent sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson el al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:
  • Gap extension penalty 0.05
  • Percent identity for multiple sequence can also be determined using MUSCLE (Edgar, R.C. (2004 ) Nuc. Acids Res. 32: 1792-97 and Edgar, R.C. (2004) Bioinformatics 5:113). In all cases, deletions are counted as non-identical residues, compared to a reference sequence.
  • “Fused” polypeptide sequences are connected, i.e., operably linked, via a peptide bond between two subject polypeptide sequences.
  • filamentous fungi refers to all filamentous forms of the subdivision
  • Eumycotina particulary Pezizomycotina species.
  • dry solids content refers to the total solids of a slurry in a dry weight percent basis.
  • slurry refers to an aqueous mixture containing insoluble solids.
  • SSF saccharification and fermentation
  • An“ethanol ogenic microorganism” refers to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol.
  • the term“fermented beverage” refers to any beverage produced by a method comprising a fermentation process, such as a microbial fermentation, e.g ., a bacterial and/or fungal fermentation.
  • a fermentation process such as a microbial fermentation, e.g ., a bacterial and/or fungal fermentation.
  • “Beer” is an example of such a fermented beverage, and the term“beer” is meant to comprise any fermented wort produced by fermentation/brewing of a starch-containing plant material. Often, beer is produced exclusively from malt or adjunct, or any combination of malt and adjunct.
  • malt refers to any malted cereal grain, such as malted barley or wheat.
  • adjuncts refers to any starch and/or sugar containing plant material that is not malt, such as barley or wheat malt.
  • adjuncts include common corn grits, refined corn grits, brewer’s milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, cassava and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like.
  • the term“mash” refers to an aqueous slurry of any starch and/or sugar containing plant material, such as grist, e.g. , comprising crushed barley malt, crushed barley, and/or other adjunct or a combination thereof, mixed with water later to be separated into wort and spent grains.
  • grist e.g. , comprising crushed barley malt, crushed barley, and/or other adjunct or a combination thereof, mixed with water later to be separated into wort and spent grains.
  • wort refers to the unfermented liquor run-off following extracting the grist during mashing.
  • One way to increase the activity of an a-amylase is to reduce the p/fa of its general acid. Lowering the p K & of a general acid results in an increase in reactivity of the protonated species.
  • the Bronsted equation for the reactivity of a general acid see, e.g., Jencks, W.P.
  • the model a-amylase used to exemplify the present compositions and methods is an a- amylase from a Bacillus sp., herein refered to as“BspAmy24.”
  • the amino acid sequence of BspAmy24 a-amylase is shown, below, as SEQ ID NO: 1 :
  • BspAmy24 is similar to an a-amylase from a Bacillus sp. 707 that is refered to as “Amy707” a-amylase.
  • the amino acid sequence of Amy707 a-amylase is shown, below, as SEQ ID NO: 2:
  • BspAmy24 is also similar to an a-amylase from another Bacillus sp. that is referred to as AA560 a-amylase, having an amino acid sequence as shown below, as SEQ ID NO: 3: HHNGTNGTMM QYFEWYLPND GNHWNRLRSD ASNLKDKGIS AVWIPPAWKG
  • the variant a-amylase has at least 60%, at least 70%, at least 80%, at least 85%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity to SEQ ID NO: 1, 2 and/or 3, excluding the wild-type BspAmy24, Amy707 and AA560, and known variants, thereof.
  • a-amylases It is known that many bacterial (and other) a-amylases share the same fold, and often benefit from the same mutations. In the present case, corresponding amino acid positions in other a-amylases can readily be identified by amino acid sequence alignment with BspAmy24, Amy707 and AA560 using Clustal W with default parameters a-amylases in which the foregoing mutations are likely to produce a performance benefit include those having a similar fold and/or having 60% or greater amino acid sequence identity to any of the well-known Bacillus amylases (e.g., from B. lichenifomis, B. stearothermophilus, B. amyloliquifaciens, Bacillus sp.
  • Bacillus amylases e.g., from B. lichenifomis, B. stearothermophilus, B. amyloliquifaciens, Bacillus sp.
  • mutations including substitutions, insertions, and deletions, that produce a beneficial effect in BspAmy24 are particularly likely to produce a similar effect in Amy707 and AA560 a-amylase, and vice versa.
  • the present amylases further include one or more mutations that provide a further performance or stability benefit.
  • Exemplary performance benfits include but are not limited to increased hydrolysis of a starch substrate, increased grain, cereal or other starch substrate liquifaction performance, increased cleaning performance, increased thermal stability, increased storage stability, increased solubility, an altered pH profile, decreased calcium dependence, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, and increased expression.
  • the performance benefit is realized at a relatively low temperature. In some cases, the performance benefit is realized at relatively high temperature.
  • the present a-amylase variants additionally have at least one mutation in the calcium binding loop based on the work of Suzuki et al. (1989) J. Biol. Chem. 264: 18933-938.
  • Exemplary mutations include a deletion or substitution at one or more residues corresponding to positions 181, 182, 183 and/or 184 in any of SEQ ID NOs: 1-3.
  • the mutation corresponds to the deletion of 181 and 182 or 183 and 184 (using any of SEQ ID NOs: 1-3 for numbering).
  • Homologous residues in other amylases can be determined by structural alignment, or by primary structure alignment.
  • the present a-amylase variants additionally have at least one mutation known to produce a performance, stability, or solubility benefit in other microbial a- amylases, including but not limited to those having a similar fold and/or having 60% or greater amino acid sequence identity to any of SEQ ID NOs: 1-3, Carbohydrate-Active Enzymes database (CAZy) Family 13 amylases, or any amylase that has heretofore been referred to by the descriptive term,“Termamyl-like.” Amino acid sequence identity can be determined using Clustal W with default parameters.
  • the present amylases may include any number of conservative amino acid substitutions. Exemplary conservative amino acid substitutions are listed in Table 2.
  • the present amylase may also be derived from any of the above-described amylase variants by substitution, deletion or addition of one or several amino acids in the amino acid sequence, for example less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or even less than 2 substitutions, deletions or additions.
  • Such variants should have the same activity as amylase from which they were derived.
  • Particular deletions include N-terminal and/or C-terminal truncations of one or a few amino acid residues, for example, 1, 2, 3, 4, or 5 amino acid residues.
  • the present amylase may be“precursor,”“immature,” or“full-length,” in which case they include a signal sequence, or“mature,” in which case they lack a signal sequence. Mature forms of the polypeptides are generally the most useful. Unless otherwise noted, the amino acid residue numbering used herein refers to the mature forms of the respective amylase polypeptides. The present amylase polypeptides may also be truncated to remove the N or C- termini, so long as the resulting polypeptides retain amylase activity.
  • the present amylase may be a“chimeric,”“hybrid” or“domain swap” polypeptide, in that it includes at least a portion of a first amylase polypeptide, and at least a portion of a second amylase polypeptide.
  • the present amylases may further include heterologous signal sequence, an epitope to allow tracking or purification, or the like.
  • Exemplary heterologous signal sequences are from B. licheniformis amylase (LAT), B. subtilis (AmyE or AprE), and
  • nucleic acids encoding a variant amylase polypeptide are provided.
  • the nucleic acid may encode a particular amylase polypeptide, or an amylase having a specified degree of amino acid sequence identity to the particular amylase.
  • the nucleic acid encodes an amylase having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity to one or more of SEQ ID NOs: 1-3. It will be appreciated that due to the degeneracy of the genetic code, a plurality of nucleic acids may encode the same polypeptide.
  • the present variant amylases can be produced in host cells, for example, by secretion or intracellular expression, using methods well-known in the art. Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used to prepare a concentrated, variant-a-amylase-polypeptide-containing solution.
  • variant a-amylase polypeptides can be enriched or partially purified as generally described above by removing cells via flocculation with polymers.
  • the enzyme can be enriched or purified by microfiltration followed by
  • the enzyme does not need to be enriched or purified, and whole broth culture can be lysed and used without further treatment. The enzyme can then be processed, for example, into granules. 4. Carbohydrate processing compositions and uses of variant amylases
  • Variants amylases are useful for a variety of carbohydrate processing applications that are well-known in the art and are not reiterated, herein. These uses include fuel ethanol production, syrup production and the production of other valuable biochemicals.
  • Useful starch substrates may be obtained from, e.g ., tubers, roots, stems, legumes, cereals or whole grain. More specifically, the granular starch may be obtained from com, cobs, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, peas, bean, banana, or potatoes.
  • Specifically contemplated starch substrates are corn starch and wheat starch.
  • the starch from a grain may be ground or whole and includes com solids, such as kernels, bran and/or cobs.
  • the starch may also be highly refined raw starch or feedstock from starch refinery processes.
  • Gelatinization is generally performed simultaneously with, or followed by, contacting a starch substrate with an a-amylase, although additional liquefaction-inducing enzymes optionally may be added.
  • the starch substrate prepared as described above is slurried with water. Liquifaction may also be performed at or below the liquifaction tempratures, as in a“cold cook” or“no cook process.”
  • the liquefied starch can be saccharified into a syrup that is rich in lower DP (e.g., DP1 + DP2) saccharides, using variant amylases, optionally in the presence of another enzyme(s).
  • DP e.g., DP1 + DP2
  • variant amylases optionally in the presence of another enzyme(s).
  • the exact composition of the products of saccharification depends on the combination of enzymes used, as well as the type of granular starch processed. Saccharification and fermentation may be performed simultaneously or in an overlapping manner (see, below).
  • the soluble starch hydrolysate produced by treatment with amylase can be converted into high fructose starch-based syrup (HFSS), such as high fructose com syrup (HFCS).
  • HFSS high fructose starch-based syrup
  • This conversion can be achieved using a glucose isomerase, particularly a glucose isomerase immobilized on a solid support.
  • the soluble starch hydrolysate can be fermented by contacting the starch hydrolysate with a fermenting organism.
  • EOF products include metabolites, such as citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, itaconic acid and other carboxylic acids, glucono delta-lactone, sodium erythorbate, lysine and other amino acids, omega 3 fatty acid, butanol, isoprene, 1,3 -propanediol and other biomaterials.
  • Ethanologenic microorganisms include yeast, such as Saccharomyces cerevisiae and bacteria, such as Zymomonas moblis , expressing alcohol dehydrogenase and pyruvate decarboxylase. Improved strains of ethanologenic microorganisms are known in the art.
  • yeast Commercial sources include ETHANOL RED® (LeSaffre); FERMAXTM (Martrex), THERMOSACC®, TRANSFERM® Yield+ and YP3TM (Lallemand); RED STAR® (Red Star); FERMIOL® (DSM Specialties); SUPERSTART® (Alltech); and SYNERXIA® and
  • compositions comprising variants amylases and additional enzymes
  • Variant amylases may be combined with a glucoamylase (EC 3.2.1.3), from e.g., Trichoderma, Aspergillus , Talaromyces, Clostridium , Fusarium , Thielavia , Thermomyces , Athelia , Humicola , Penicillium , Artomyces, Gloeophyllum , Pycnoporus, Steccherinum , Trametes etc.
  • Suitable commercial glucoamylases include AMG 200L; AMG 300 L; SANTM SUPER and AMGTM E (Novozymes); OPTIDEX® 300 and OPTIDEX L-400 (Danisco US Inc.);
  • AMIGASETM and AMIGASETM PLUS DSM
  • G-ZYME® G900 Enzyme Bio-Systems
  • G-ZYME® G990 ZR G-ZYME® G990 ZR.
  • amylase Other suitable enzymes that can be used with amylase include phytase, protease, pullulanase, b-amylase, isoamylase, a-glucosidase, cellulase, xylanase, other hemicellulases, b- glucosidase, transferase, pectinase, lipase, cutinase, esterase, mannanase, redox enzymes, a different a-amylase, or a combination thereof.
  • compositions comprising the present amylases may be aqueous or non-aqueous formulations, granules, powders, gels, slurries, pastes, etc., which may further comprise any one or more of the additional enzymes listed, herein, along with buffers, salts, preservatives, water, co-solvents, surfactants, and the like.
  • Such compositions may work in combination with endogenous enzymes or other ingredients already present in a slurry, water bath, washing machine, food or drink product, etc., for example, endogenous plant (including algal) enzymes, residual enzymes from a prior processing step, and the like.
  • the present invention also relates to a“food composition,” including but not limited to a food product, animal feed and/or food/feed additives, comprising an amylase, and methods for preparing such a food composition comprising mixing variant amylase with one or more food ingredients, or uses thereof. Furthermore, the present invention relates to the use of an amylase in the preparation of a food composition, wherein the food composition is baked subsequent to the addition of the polypeptide of the invention.
  • the present variant amylase may be a component of a brewing composition used in a process of brewing, /. e. , making a fermented malt beverage.
  • Non-fermentable carbohydrates form the majority of the dissolved solids in the final beer. This residue remains because of the inability of malt amylases to hydrolyze the alpha- 1, 6-linkages of the starch.
  • the non- fermentable carbohydrates contribute about 50 calories per 12 ounces of beer.
  • compositions and methods of treating fabrics e.g ., to desize a textile
  • an amylase e.g., to desize a textile
  • Fabric-treating methods are well known in the art (see, e.g., U.S. Patent No. 6,077,316).
  • the feel and appearance of a fabric can be improved by a method comprising contacting the fabric with an amylase in a solution.
  • the fabric can be treated with the solution under pressure.
  • An aspect of the present compositions and methods is a cleaning composition that includes an amylase as a component.
  • An amylase polypeptide can be used as a component in detergent compositions for, e.g, hand washing, laundry washing, dishwashing, and other hard- surface cleaning.
  • Such compositions include heavy duty liquid (HDL), heavy duty dry (HDD), and hand (manual) laundry detergent compositions, including unit dose format laundry detergent compositions, and automatic dishwashing (ADW) and hand (manual) dishwashing compositions, including unit dose format dishwashing compositions.
  • An amylase polypeptide may be a component of a detergent composition, as the only enzyme or with other enzymes including other amylolytic enzymes. As such, it may be included in the detergent composition in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme.
  • the detergent composition may be in any useful form, e.g ., as powders, granules, pastes, bars, or liquid.
  • a liquid detergent may be aqueous, typically containing up to about 70% of water and 0% to about 30% of organic solvent. It may also be in the form of a compact gel type containing only about 30% water.
  • the detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic.
  • the detergent composition may additionally comprise one or more other enzymes, such as proteases, another amylolytic enzyme, mannanase, cutinase, lipase, cellulase, pectate lyase, perhydrolase, xylanase, peroxidase, and/or laccase in any combination.
  • enzymes such as proteases, another amylolytic enzyme, mannanase, cutinase, lipase, cellulase, pectate lyase, perhydrolase, xylanase, peroxidase, and/or laccase in any combination.
  • detergent compositions for inclusion of the present a-amylase are described, below. Many of these composition can be provided in unit dose format for ease of use. Unit dose formulations and packaging are described in, for example, US20090209445A1, US20100081598 Al, US7001878B2, EP1504994B1, W02001085888A2, W02003089562A1, W02009098659A1, W02009098660A1, W02009112992A1, W02009124160A1,
  • Exemplary HDL laundry detergent compositions includes a detersive surfactant (l0%-40% wt/wt), including an anionic detersive surfactant (selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and/or mixtures thereof), and optionally non-ionic surfactant (selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohol, for example a C8-C18 alkyl ethoxylated alcohol and/or C6-C12 alkyl phenol alkoxylates), wherein the weight ratio of anionic detersive surfactant (with a hydrophilic index (HIc) of from 6.0 to 9) to non-ionic detersive
  • Suitable detersive surfactants also include cationic detersive surfactants (selected from a group of alkyl pyridinium compounds, alkyl quarternary ammonium compounds, alkyl quarternary phosphonium compounds, alkyl ternary sulphonium compounds, and/or mixtures thereof); zwitterionic and/or amphoteric detersive surfactants (selected from a group of alkanolamine sulpho-betaines); ampholytic surfactants; semi-polar non-ionic surfactants and mixtures thereof.
  • the composition may optionally include, a surfactancy boosting polymer consisting of amphiphilic alkoxylated grease cleaning polymers (selected from a group of alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkylenimines in the range of 0.05 wt%-l0 wt%) and/or random graft polymers (typically comprising of hydrophilic backbone comprising monomers selected from the group consisting of: unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyalcohols such as glycerol, and mixtures thereof; and hydrophobic side chain(s) selected from the group consisting of: C4-C25 alkyl group, polypropylene, polybutylene, vinyl ester of a saturated C1-C6 mono-carboxylic acid, C1-C6 alky
  • the composition may include additional polymers such as soil release polymers (include anionically end-capped polyesters, for example SRP1, polymers comprising at least one monomer unit selected from saccharide, dicarboxylic acid, polyol and combinations thereof, in random or block configuration, ethylene terephthalate-based polymers and co-polymers thereof in random or block configuration, for example Repel-o-tex SF, SF-2 and SRP6, Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, Marloquest SL), anti redeposition polymers (0.1 wt% to 10 wt%, include carboxylate polymers, such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof, vinylpyrrolidone homopoly
  • the composition may further include saturated or unsaturated fatty acid, preferably saturated or unsaturated C12-C24 fatty acid (0 wt% to 10 wt%); deposition aids (examples for which include polysaccharides, preferably cellulosic polymers, poly diallyl dimethyl ammonium halides (DADMAC), and co-polymers of DAD MAC with vinyl pyrrolidone, acrylamides, imidazoles, imidazolinium halides, and mixtures thereof, in random or block configuration, cationic guar gum, cationic cellulose such as cationic hydoxyethyl cellulose, cationic starch, cationic polyacylamides, and mixtures thereof.
  • deposition aids include polysaccharides, preferably cellulosic polymers, poly diallyl dimethyl ammonium halides (DADMAC), and co-polymers of DAD MAC with vinyl pyrrolidone, acrylamides, imidazo
  • the composition may further include dye transfer inhibiting agents, examples of which include manganese phthalocyanine, peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles and/or mixtures thereof; chelating agents, examples of which include ethylene-diamine-tetraacetic acid (EDTA), diethylene triamine penta methylene phosphonic acid (DTPMP), hydroxy-ethane diphosphonic acid (HEDP),
  • dye transfer inhibiting agents examples of which include manganese phthalocyanine, peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles and/or mixtures thereof
  • ethylenediamine N,N'-disuccinic acid ethylenediamine N,N'-disuccinic acid
  • MGDA methyl glycine diacetic acid
  • DTP A diethylene triamine penta acetic acid
  • HPNO propylene diamine tetracetic acid
  • MGDA methyl glycine diacetic acid
  • glutamic acid N,N-diacetic acid N,N-dicarboxymethyl glutamic acid tetrasodium salt (GLDA)
  • NTA nitrilotriacetic acid
  • NTA 4,5-dihydroxy-m-benzenedisulfonic acid
  • citric acid and any salts thereof N- hydroxyethylethylenediaminetri-acetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropi
  • the composition preferably included enzymes (generally about 0.01 wt% active enzyme to 0.03 wt% active enzyme) selected from proteases, amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and any mixture thereof.
  • enzymes generally about 0.01 wt% active enzyme to 0.03 wt% active enzyme selected from proteases, amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferases, perhydrolases, arylesterases, and any mixture thereof.
  • the composition may include an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g ., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
  • an enzyme stabilizer examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g ., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
  • the composition optionally includes silicone or fatty-acid based suds suppressors; hueing dyes, calcium and magnesium cations, visual signaling ingredients, anti -foam (0.001 wt% to about 4.0wt%), and/or structurant/thickener (0.01 wt% to 5 wt%, selected from the group consisting of diglycerides and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose based materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof).
  • silicone or fatty-acid based suds suppressors hueing dyes, calcium and magnesium cations, visual signaling ingredients, anti -foam (0.001 wt% to about 4.0wt%), and/or structurant/thickener (0.01 wt% to 5 wt%, selected from the group consisting of diglycerides and triglycerides, ethylene glycol distearate, microcrystalline
  • the composition can be any liquid form, for example a liquid or gel form, or any combination thereof.
  • the composition may be in any unit dose form, for example a pouch.
  • Exemplary HDD laundry detergent compositions includes a detersive surfactant, including anionic detersive surfactants (e.g ., linear or branched or random chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and/or mixtures thereof), non-ionic detersive surfactant (e.g., linear or branched or random chain, substituted or unsubstituted C8-C18 alkyl ethoxylates, and/or C6-C12 alkyl phenol alkoxylates), cationic detersive surfactants (e.g, alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium
  • anionic detersive surfactants e.g ., linear or branched or random chain, substitute
  • zwitterionic and/or amphoteric detersive surfactants e.g, alkanolamine sulpho-betaines
  • ampholytic surfactants e.g, ampholytic surfactants, semi-polar non-ionic surfactants, and mixtures thereof
  • builders including phosphate free builders for example zeolite builders examples which include zeolite A, zeolite X, zeolite P and zeolite MAP in the range of 0 wt% to less than 10 wt%), phosphate builders (for example sodium tri-polyphosphate in the range of 0 wt% to less than 10 wt%), citric acid, citrate salts and nitrilotriacetic acid, silicate salt (e.g, sodium or potassium silicate or sodium meta-silicate in the range of 0 wt% to less than 10 wt%, or layered silicate (SKS-6)); carbonate salt (e.g, sodium carbonate salt (e.g,
  • the composition preferably includes enzymes, e.g., proteases, amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferase, perhydrolase, arylesterase, and any mixture thereof.
  • enzymes e.g., proteases, amylases, lipases, cellulases, choline oxidases, peroxidases/oxidases, pectate lyases, mannanases, cutinases, laccases, phospholipases, lysophospholipases, acyltransferase, perhydrolase, arylesterase, and any mixture thereof.
  • the composition may optionally include additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accord, hueing agents, additional polymers, including fabric integrity and cationic polymers, dye-lock ingredients, fabric- softening agents, brighteners (for example C.I. Fluorescent brighteners), flocculating agents, chelating agents, alkoxylated polyamines, fabric deposition aids, and/or cyclodextrin.
  • additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accord, hueing agents, additional polymers, including fabric integrity and cationic polymers, dye-lock ingredients, fabric- softening agents, brighteners (for example C.I. Fluorescent brighteners), flocculating agents, chelating agents, alkoxylated polyamines, fabric deposition aids, and/or cyclodextrin.
  • Exemplary ADW detergent composition includes non-ionic surfactants, including ethoxylated non-ionic surfactants, alcohol alkoxylated surfactants, epoxy-capped
  • organic bleach e.g., organic peroxyacids, including diacyl and tetraacylperoxides, especially diperoxydodecanedioc acid, diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid
  • bleach activators i.e., organic peracid precursors in the range from about 0.1 % to about 10% by weight
  • bleach catalysts e.g, manganese triazacyclononane and related complexes, Co, Cu, Mn, and Fe bispyridylamine and related complexes, and pentamine acetate cobalt(III) and related complexes
  • metal care agents in the range from about 0.1% to 5% by weight (e.g, benzatriazoles, metal salts and complexes, and/or silicates); enzymes in the range from about 0.01 to 5.0 mg of active enzyme per gram of
  • any of the cleaning compositions described, herein, may include any number of additional enzymes.
  • the enzyme(s) should be compatible with the selected detergent, (e.g, with respect to pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, and the like), and the enzyme(s) should be present in effective amounts.
  • the following enzymes are provided as examples.
  • Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are included, as well as naturally processed proteins.
  • the protease may be a serine protease or a metalloprotease, an alkaline microbial protease, a trypsin-like protease, or a chymotrypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (see, e.g, WO 89/06279).
  • Exemplary proteases include but are not limited to those described in W095/23221, WO 92/21760, W02008010925, WO20100566356, WO2011072099, WO201113022, WO2011140364, WO 2012151534, WO2015038792, WO2015089441, WO2015089447, WO2015143360, W02016001449, W02016001450, WO2016061438, W020l6069544,W020l6069548, WO2016069552, WO 2016069557, WO2016069563, WO2016069569, WO2016087617, WO2016087619, WO2016145428, WO2016174234, WO2016183509, WO2016202835, WO2016205755, US 2008/0090747, US 5,801,039, US 5,340,735, US 5,500,364, US 5,855,625, RE 34,606, US 5,955,340, US 5,700
  • Exemplary commercial proteases include, but are not limited to MAXATASE, MAXACAL, MAXAPEM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX®, EXCELLASE®, PREFERENZTM proteases (e g. P100, P110, P280), EFFECTENZTM proteases (e.g. P1000, P1050, P2000), EXCELLENZTM proteases (e g. P1000), ULTIMASE®, and PURAFAST (Danisco US); ALCALASE®, ALCALASE® ULTRA, BLAZE®, BLAZE® EVITY®, BLAZE® EVITY® 16L, CORONASE®,
  • Suitable proteases include naturally occurring proteases or engineered variants specifically selected or engineered to work at relatively low temperatures.
  • Suitable lipases include those of bacterial or fungal origin. Chemically modified, proteolytically modified, or protein engineered mutants are included. Examples of useful lipases include but are not limited to lipases from Humicola (synonym Thermomyces ), e.g. , from H. lanuginosa (I lanuginosus) (see e.g., EP 258068 and EP 305216), from H. insol ens (see e.g., WO 96/13580); a Pseudomonas lipase (e.g, from P. alcaligenes or P. pseudoalcaligenes, see, e.g., EP 218 272), P.
  • Humicola semomyces
  • H. lanuginosa I lanuginosus
  • Pseudomonas lipase e.g, from P. alcaligenes or P. pseudoalcaligenes, see, e.g.,
  • cepacia see e.g., EP 331 376
  • P. stutzeri see e.g., GB 1,372,034
  • P. fluorescens Pseudomonas sp. strain SD 705 (see e.g., WO 95/06720 and WO 96/27002)
  • P. wisconsinensis see e.g., WO 96/12012
  • Bacillus lipase e.g., from B. subtilis; see e.g. , Dartois et al. (1993) Biochemica et Biophysica Acta 1131 :253-360
  • B. subtilis see e.g., Dartois et al. (1993) Biochemica et Biophysica Acta 1131 :253-360
  • stearothermophilus see e.g., JP 64/744992
  • B. pumilus see e.g, WO 91/16422.
  • Additional lipase variants contemplated for use in the formulations include those described for example in: WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615,
  • Exemplary commercial lipases include, but are not limited to Ml LIPASE, LETMA FAST, and LIPOMAX (Genencor); LIPEX®, LIPOCLEAN®, LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE P (Amano Pharmaceutical Co. Ltd).
  • polyesterases can be included in the composition, such as those described in, for example, WO 01/34899, WO 01/14629, and US6933140.
  • compositions can be combined with other amylases, including other a- amylases. Such a combination is particularly desirable when different a-amylases demonstrate different performance characteristics and the combination of a plurality of different a-amylases results in a composition that provides the benefits of the different a-amylases.
  • Other amylases include commercially available amylases, such as but not limited to STAINZYME®,
  • NATALASE®, DURAMYL®, TERMAMYL®, FUNGAMYL® and BANTM Novo Nordisk A/S and Novozymes A/S
  • RAPIDASE®, POWERASE®, PURASTAR® and PREFERENZTM (from DuPont Industrial Biosciences).
  • Exemplary a-amylases are described in W09418314A1, US20080293607, W02013063460, WO10115028, W02009061380A2, WO2014099523, WO2015077126A1, WO2013184577, WO2014164777, W09510603, W09526397,
  • WO02092797 WO0166712, W00188107, WO0196537, W00210355, W02006002643, W02004055178, and W09813481.
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas , Humicola , Fusarium, Thielavia , Acremonium , e.g., the fungal cellulases produced from Humicola insolens , Myceliophthora thermophila and Fusarium oxysporum disclosed for example in U.S. Patent Nos. 4,435,307; 5,648,263; 5,691,178;
  • Exemplary cellulases contemplated for use are those having color care benefit for the textile.
  • Examples of such cellulases are cellulases described in for example EP 0495257, EP 0531372, WO 96/11262, WO 96/29397, and WO 98/08940.
  • Other examples are cellulase variants, such as those described in WO 94/07998; WO 98/12307; WO 95/24471; PCT/DK98/00299; EP 531315; U.S. Patent Nos. 5,457,046; 5,686,593; and
  • Exemplary cellulases include those described in W02005054475, W02005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and US Provisional Appl. Nos. 62/296,678 and 62/435340.
  • Exemplary commercial cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®, CAREZYME®, CAREZYME® PREMIUM, ENDOLASE®, and RENOZYME® (Novozymes); REVITALENZ®lOO, REVITALENZ® 200/220 and REVITALENZ® 2000 (Danisco US); and KAC-500(B) (Kao Corporation).
  • Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as, for example, as is described in W02016007929; USPNs 6,566,114, 6,602,842, and 6,440,991; and International Appl. Nos. PCT/US2016/060850 and PCT/US2016/060844.
  • Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as, for example, as is described in W02016007929; USPNs 6566114, 6,602,842, and 6,440,991; and International Appl. Nos. PCT/US2016/060850 and PCT/US2016/060844.
  • Suitable peroxidases/oxidases contemplated for use in the compositions include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g ., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include for example GUARDZYMETM (Novo Nordisk A/S and Novozymes A/S).
  • the detergent composition can also comprise 2,6-P-D-fructan hydrolase, which is effective for removal/cleaning of biofilm present on household and/or industrial textile/laundry.
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive i.e. a separate additive or a combined additive, can be formulated, e.g. , as a granulate, a liquid, a slurry, and the like.
  • Exemplary detergent additive formulations include but are not limited to granulates, in particular non dusting granulates, liquids, in particular stabilized liquids or slurries.
  • the detergent composition may be in any convenient form, e.g. , a bar, a tablet, a powder, a granule, a paste, or a liquid.
  • a liquid detergent may be aqueous, typically containing up to about 70% water, and 0% to about 30% organic solvent. Compact detergent gels containing about 30% or less water are also contemplated.
  • W02013063460 Numerous exemplary detergent formulations to which the present amylases can be added (or is in some cases are identified as a component of) are described in W02013063460. These include commercially available unit dose detergent formulations/packages such as PUREX® UltraPacks (Henkel), FINISH® Quantum (Reckitt Benckiser), CLOROXTM 2 Packs (Clorox), OxiClean Max Force Power Paks (Church & Dwight), TIDE® Stain Release,
  • a homology model of BspAmy24 was constructed as follows.
  • the amino acid sequence of BspAmy24 (SEQ ID NO: 1) was used as a query in MOE (Chemical Computing Group, Montreal, CA) to search the public structure database.
  • MOE Chemical Computing Group, Montreal, CA
  • the Bacillus licheniformis alpha- amylase (1BLI) was the top public hit.
  • The“homology model” function with all default parameters was used to create a model.
  • An X-ray diffraction crystal structure was also determined for a BspAmy24 variant; this experimental structure closely matched the homology model and supported the analysis performed with the homology model.
  • BspAmy24-Vl (having a a deletion of residues R181 and G182) and variants of BspAmy24-Vl were expressed in Bacillus licheniformis cells after following standard cloning procedures to introduce appropriate DNA sequences. Cells were grown for 68 hours in expression medium suitable for secreted protein expression from B. licheniformis. Secreted protein was harvested by centrifugation followed by filtration through 0.45 pm membranes (EMD Millipore). In some cases, additional purification was performed using ion exchange chromatography with Phenyl Sepharose 6 Fast Flow resin (GE Healthcare). Protein
  • the activity of the a-amylases at different pH values was measured using a microswatch assay.
  • a microswatch assay In a 96-well microtiter plate containing two micro-swatches (CS28 starch stain with dye on cotton, Center for Testmaterials, Vlaardingen, Netherlands), diluted enzyme was added to buffer with 2 mM CaCb and 0.005% Tween-80, total 200 m ⁇ reaction volume. The plate was incubated at 50°C for 15 minutes with shaking at 1,150 rpm. The supernatant was then removed from the swatch plate and the amount of dye released into the supernatant was quantified optically. The release of dye into the supernatant correlated with enzyme activity.
  • the rate constant was determined at each pH and the plot of the log of the rate constant versus pH was fit to a single or double ionization model, as follows, where & max represents the maximum rate constant, p K a , p/fai, and pK di represent p K a values for the enzyme, and pH represents the pH of the reaction:
  • Example 2 Results obtained using a-amylase variants
  • the activity was measured at pH 8.5 and pH 10.5 and the ratio of the activity at pH 8.5 compared to the activity at pH 10.5 was determined according to the formula (“WT” in this context is the del-Rl8l-Gl82 molecule):

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Abstract

L'invention concerne des compositions et des procédés se rapportant à des variants d'alpha-amylases. Les variants d'alpha-amylases sont utiles, par exemple, pour la liquéfaction et la saccharification de l'amidon, le nettoyage de taches d'amidon, le désencollage textile, la cuisson et le brassage.
PCT/US2019/044254 2018-07-31 2019-07-30 Variants d'alpha-amylases ayant des substitutions d'acides aminés qui abaissent le pka de l'acide général Ceased WO2020028443A1 (fr)

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US17/264,531 US12584119B2 (en) 2018-07-31 2019-07-30 Variant alpha-amylases having amino acid substitutions that lower the PKA of the general acid
JP2021505722A JP7530884B2 (ja) 2018-07-31 2019-07-30 一般酸のｐｋａを低下させるアミノ酸置換を有する変異体アルファ－アミラーゼ
MX2021001213A MX2021001213A (es) 2018-07-31 2019-07-30 Variantes de alfa-amilasas que tienen sustituciones de aminoácidos que bajan el pka del ácido general.
CA3108284A CA3108284A1 (fr) 2018-07-31 2019-07-30 Variants d'alpha-amylases ayant des substitutions d'acides amines qui abaissent le pka de l'acide general
EP19758538.3A EP3830231A1 (fr) 2018-07-31 2019-07-30 Variants d'alpha-amylases ayant des substitutions d'acides aminés qui abaissent le pka de l'acide général
CN201980064393.3A CN112805361A (zh) 2018-07-31 2019-07-30 具有降低广义酸的PKA的氨基酸取代的变体α-淀粉酶

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WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

Citations (152)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1372034A (en) 1970-12-31 1974-10-30 Unilever Ltd Detergent compositions
US4435307A (en) 1980-04-30 1984-03-06 Novo Industri A/S Detergent cellulase
EP0218272A1 (fr) 1985-08-09 1987-04-15 Gist-Brocades N.V. Enzymes lipolytiques et leur usage dans des compositions détergentes
EP0258068A2 (fr) 1986-08-29 1988-03-02 Novo Nordisk A/S Additif enzymatique pour détergent
EP0260105A2 (fr) 1986-09-09 1988-03-16 Genencor, Inc. Préparation d'enzymes à activité modifiée
EP0305216A1 (fr) 1987-08-28 1989-03-01 Novo Nordisk A/S Lipase recombinante de humicola et procédé de production de lipases recombinantes de humicola
WO1989006279A1 (fr) 1988-01-07 1989-07-13 Novo-Nordisk A/S Genes de subtilisine mutes
EP0331376A2 (fr) 1988-02-28 1989-09-06 Amano Pharmaceutical Co., Ltd. ADN recombinant, bactérie du genre pseudomonas le contenant et son utilisation dans un procédé de production de lipase
WO1989009259A1 (fr) 1988-03-24 1989-10-05 Novo-Nordisk A/S Preparation de cellulase
EP0407225A1 (fr) 1989-07-07 1991-01-09 Unilever Plc Enzymes et compositions détergentes enzymatiques
WO1991016422A1 (fr) 1990-04-14 1991-10-31 Kali-Chemie Aktiengesellschaft Lipases bacillaires alcalines, sequences d'adn de codage pour celles-ci et bacilles produisant ces lipases
WO1992005249A1 (fr) 1990-09-13 1992-04-02 Novo Nordisk A/S Variantes lipasiques
EP0495257A1 (fr) 1991-01-16 1992-07-22 The Procter & Gamble Company Compositions de détergent compactes contenant de la cellulase de haute activité
WO1992021760A1 (fr) 1991-05-29 1992-12-10 Cognis, Inc. Enzymes proteolytiques mutantes tirees de bacillus
EP0531372A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Preparation de cellulase comprenant un enzyme d'endoglucanase.
EP0531315A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Enzyme capable de degrader la cellulose ou l"hemicellulose.
WO1993024618A1 (fr) 1992-06-01 1993-12-09 Novo Nordisk A/S Variante de peroxydase avec stabilite amelioree vis-a-vis du peroxyde d'hydrogene
WO1994001541A1 (fr) 1992-07-06 1994-01-20 Novo Nordisk A/S Lipase de c. antarctica et variantes lipasiques
WO1994007998A1 (fr) 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase
USRE34606E (en) 1984-05-29 1994-05-10 Genencor, Inc. Modified enzymes and methods for making same
WO1994018314A1 (fr) 1993-02-11 1994-08-18 Genencor International, Inc. Alpha-amylase stable a l'oxydation
WO1994025578A1 (fr) 1993-04-27 1994-11-10 Gist-Brocades N.V. Nouveaux variants de lipase utilises dans des detergents
WO1995006720A1 (fr) 1993-08-30 1995-03-09 Showa Denko K.K. Nouvelle lipase, micro-organisme la produisant, procede de production de cette lipase, et utilisation de ladite lipase
WO1995010602A1 (fr) 1993-10-13 1995-04-20 Novo Nordisk A/S Variants de peroxydase stables par rapport a h2o¿2?
WO1995010603A1 (fr) 1993-10-08 1995-04-20 Novo Nordisk A/S Variants d'amylase
WO1995014783A1 (fr) 1993-11-24 1995-06-01 Showa Denko K.K. Gene de lipase et lipase variante
WO1995022615A1 (fr) 1994-02-22 1995-08-24 Novo Nordisk A/S Procede pour preparer un variant d'une enzyme lipolytique
WO1995023221A1 (fr) 1994-02-24 1995-08-31 Cognis, Inc. Enzymes ameliorees et detergents les contenant
WO1995024471A1 (fr) 1994-03-08 1995-09-14 Novo Nordisk A/S Nouvelles cellulases alcalines
WO1995026397A1 (fr) 1994-03-29 1995-10-05 Novo Nordisk A/S Amylase alcaline issue d'un bacille
WO1995030744A2 (fr) 1994-05-04 1995-11-16 Genencor International Inc. Lipases a resistance aux tensioactifs amelioree
WO1995035381A1 (fr) 1994-06-20 1995-12-28 Unilever N.V. Lipases modifiees provenant de pseudomonas et leur utilisation
WO1996000292A1 (fr) 1994-06-23 1996-01-04 Unilever N.V. Pseudomonas lipases modifiees et leur utilisation
WO1996011262A1 (fr) 1994-10-06 1996-04-18 Novo Nordisk A/S Enzyme et preparation enzymatique presentant une activite endoglucanase
WO1996012012A1 (fr) 1994-10-14 1996-04-25 Solvay S.A. Lipase, micro-organisme la produisant, procede de preparation de cette lipase et utilisation de celle-ci
WO1996013580A1 (fr) 1994-10-26 1996-05-09 Novo Nordisk A/S Enzyme a activite lipolytique
WO1996023873A1 (fr) 1995-02-03 1996-08-08 Novo Nordisk A/S Alleles d'amylase-alpha
WO1996023874A1 (fr) 1995-02-03 1996-08-08 Novo Nordisk A/S Technique de mise au point de mutants d'amylase-alpha dotes de proprietes predefinies
WO1996027002A1 (fr) 1995-02-27 1996-09-06 Novo Nordisk A/S Nouveau gene de lipase et procede de production de lipase a l'aide de celui-ci
WO1996029397A1 (fr) 1995-03-17 1996-09-26 Novo Nordisk A/S Nouvelles endoglucanases
WO1997004079A1 (fr) 1995-07-14 1997-02-06 Novo Nordisk A/S Enzyme modifiee a activite lipolytique
WO1997007202A1 (fr) 1995-08-11 1997-02-27 Novo Nordisk A/S Nouvelles enzymes lipolytiques
US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
WO1997041213A1 (fr) 1996-04-30 1997-11-06 Novo Nordisk A/S MUTANTS DUNE AMYLASE-$g(a)
US5700676A (en) 1984-05-29 1997-12-23 Genencor International Inc. Modified subtilisins having amino acid alterations
WO1998008940A1 (fr) 1996-08-26 1998-03-05 Novo Nordisk A/S Nouvelle endoglucanase
WO1998012307A1 (fr) 1996-09-17 1998-03-26 Novo Nordisk A/S Variants de cellulase
WO1998013481A1 (fr) 1996-09-26 1998-04-02 Novo Nordisk A/S Enzyme a activite amylase
WO1998015257A1 (fr) 1996-10-08 1998-04-16 Novo Nordisk A/S Derives de l'acide diaminobenzoique en tant que precurseurs de matieres tinctoriales
US5801039A (en) 1994-02-24 1998-09-01 Cognis Gesellschaft Fuer Bio Und Umwelttechnologie Mbh Enzymes for detergents
US5855625A (en) 1995-01-17 1999-01-05 Henkel Kommanditgesellschaft Auf Aktien Detergent compositions
WO1999019467A1 (fr) 1997-10-13 1999-04-22 Novo Nordisk A/S MUTANTS D'α-AMYLASE
WO1999023211A1 (fr) 1997-10-30 1999-05-14 Novo Nordisk A/S Mutants d'alpha-amylase
WO1999042567A1 (fr) 1998-02-18 1999-08-26 Novo Nordisk A/S Amylase bacillaire alcaline
WO1999046399A1 (fr) 1998-03-09 1999-09-16 Novo Nordisk A/S Preparation enzymatique de sirop de glucose a partir d'amidon
US5955340A (en) 1984-05-29 1999-09-21 Genencor International, Inc. Modified subtilisins having amino acid alterations
WO2000029560A1 (fr) 1998-11-16 2000-05-25 Novozymes A/S VARIANTES DE α-AMYLASE
US6077316A (en) 1995-07-19 2000-06-20 Novo Nordisk A/S Treatment of fabrics
WO2000060059A2 (fr) 1999-03-30 2000-10-12 NovozymesA/S Variantes d'alpha amylase
WO2000060058A2 (fr) 1999-03-31 2000-10-12 Novozymes A/S Polypeptides possedant une activite alcaline alpha-amylase et acides nucleiques codant pour ces polypeptides
WO2000060060A2 (fr) 1999-03-31 2000-10-12 Novozymes A/S Polypeptides presentant une activite alcaline alpha-amylase et acides nucleiques les codant
WO2001014532A2 (fr) 1999-08-20 2001-03-01 Novozymes A/S Amylase alcaline de bacillus
WO2001014629A1 (fr) 1999-08-20 2001-03-01 Genencor International, Inc. Modification enzymatique de la surface d'une fibre ou d'un article de polyester
WO2001034899A1 (fr) 1999-11-05 2001-05-17 Genencor International, Inc. Enzymes utiles pour changer les proprietes du polyester
WO2001064852A1 (fr) * 2000-03-03 2001-09-07 Novozymes A/S Polypeptides possedant une activite de l'alpha-amylase et acides nucleiques codant pour ces polypeptides
WO2001066712A2 (fr) 2000-03-08 2001-09-13 Novozymes A/S Variants possedant des proprietes modifiees
US6312936B1 (en) 1997-10-23 2001-11-06 Genencor International, Inc. Multiply-substituted protease variants
WO2001085888A2 (fr) 2000-05-11 2001-11-15 The Procter & Gamble Company Systeme de lessive a dosage unitise
WO2001088107A2 (fr) 2000-05-12 2001-11-22 Novozymes A/S Variantes d'alpha-amylase avec une activite 1,6 alteree
WO2001096537A2 (fr) 2000-06-14 2001-12-20 Novozymes A/S Alpha-amylase pre-oxydee
WO2002010355A2 (fr) 2000-08-01 2002-02-07 Novozymes A/S Mutants d'alpha-amylase a proprietes modifiees
US6440991B1 (en) 2000-10-02 2002-08-27 Wyeth Ethers of 7-desmethlrapamycin
WO2002092797A2 (fr) 2001-05-15 2002-11-21 Novozymes A/S Variant d'alpha-amylases ayant des proprietes modifiees
US6566114B1 (en) 1998-06-10 2003-05-20 Novozymes, A/S Mannanases
US6602842B2 (en) 1994-06-17 2003-08-05 Genencor International, Inc. Cleaning compositions containing plant cell wall degrading enzymes and their use in cleaning methods
WO2003089562A1 (fr) 2002-04-19 2003-10-30 The Procter & Gamble Company Sachets de produit de nettoyage
US6730646B1 (en) 1998-07-29 2004-05-04 Reckitt Benckiser N.V. Composition for use in a dishwasher
WO2004055178A1 (fr) 2002-12-17 2004-07-01 Novozymes A/S Alpha-amylases thermostables
EP1504994A2 (fr) 2000-11-27 2005-02-09 The Procter & Gamble Company Procédé de fabrication d'un sachet soluble dans l'eau
WO2005054475A1 (fr) 2003-12-03 2005-06-16 Meiji Seika Kaisha, Ltd. Stce d'endoglucanase et preparation de cellulase le contenant
WO2005056787A1 (fr) 2003-12-08 2005-06-23 Meiji Seika Kaisha, Ltd. Cellulase supportant les tensioactifs et procede de transformation associe
WO2006002643A2 (fr) 2004-07-05 2006-01-12 Novozymes A/S Variants d'alpha-amylases presentant des proprietes modifiees
WO2006007911A1 (fr) 2004-07-20 2006-01-26 Unilever Plc Produit de blanchisserie
US7001878B2 (en) 2003-09-22 2006-02-21 The Procter & Gamble Company Liquid unit dose detergent composition
WO2006045391A1 (fr) 2004-10-29 2006-05-04 Unilever Plc Procede de preparation d'un produit de lessive
EP1740690A1 (fr) 2004-04-28 2007-01-10 Henkel Kommanditgesellschaft auf Aktien Procede pour produire des produits detergents ou nettoyants
WO2007044993A2 (fr) 2005-10-12 2007-04-19 Genencor International, Inc. Utilisation et production d'une metalloprotease neutre stable au stockage
WO2008000567A1 (fr) 2006-06-30 2008-01-03 Unilever Plc Articles de lessive
WO2008010925A2 (fr) 2006-07-18 2008-01-24 Danisco Us, Inc., Genencor Division Variantes de protéases actives sur une large plage de températures
WO2008087426A1 (fr) 2007-01-18 2008-07-24 Reckitt Benckiser N.V. Élément de dosage et son procédé de fabrication
US7449318B2 (en) 2003-04-30 2008-11-11 Danisco A/S, Genencor Division Bacillus mHKcel cellulase
US20080293607A1 (en) 2007-03-09 2008-11-27 Jones Brian E Alkaliphilic Bacillus Species alpha-Amylase Variants, Compositions Comprising alpha-Amylase Variants, And Methods of Use
WO2009058303A2 (fr) 2007-11-01 2009-05-07 Danisco Us Inc., Genencor Division Production de thermolysine et de ses variants et utilisation dans des détergents liquides
WO2009058661A1 (fr) 2007-10-31 2009-05-07 Danisco Us Inc., Genencor Division Utilisation et production de métalloprotéases neutres stables vis-à-vis des citrates
WO2009061380A2 (fr) 2007-11-05 2009-05-14 Danisco Us Inc., Genencor Division Variants de bacillus sp. ts-23 alpha-amylase à propriétés modifiées
WO2009098660A1 (fr) 2008-02-08 2009-08-13 The Procter & Gamble Company Sachet soluble dans l'eau
WO2009098659A1 (fr) 2008-02-08 2009-08-13 The Procter & Gamble Company Procédé de fabrication d'une poche soluble dans l'eau
US20090209445A1 (en) 2006-03-22 2009-08-20 The Procter & Gamble Company Liquid treatment unitized dose composition
WO2009112992A1 (fr) 2008-03-14 2009-09-17 The Procter & Gamble Company Composition détergente pour lave-vaisselle
WO2009124160A1 (fr) 2008-04-02 2009-10-08 The Procter & Gamble Company Poche hydrosoluble renfermant une composition détergente
WO2009152031A1 (fr) 2008-06-13 2009-12-17 The Procter & Gamble Company Poche multi-compartiments
US20100081598A1 (en) 2000-11-27 2010-04-01 The Procter & Gamble Company Dishwashing method
WO2010059483A1 (fr) 2008-11-20 2010-05-27 The Procter & Gamble Company Produits nettoyants
WO2010088112A1 (fr) 2009-01-28 2010-08-05 The Procter & Gamble Company Composition de poche à plusieurs compartiments pour lessive
WO2010090915A1 (fr) 2009-02-09 2010-08-12 The Procter & Gamble Company Composition détergente
WO2010115028A2 (fr) 2009-04-01 2010-10-07 Danisco Us Inc. Système de lavage comprenant une alpha-amylase et une protéase
WO2010116139A1 (fr) 2009-04-09 2010-10-14 Reckitt Benckiser N.V. Composition détergente
WO2010135238A1 (fr) 2009-05-19 2010-11-25 The Procter & Gamble Company Procédé d'impression d'un film soluble dans l'eau
WO2011013022A1 (fr) 2009-07-28 2011-02-03 Koninklijke Philips Electronics N.V. Unité de lavage et de stérilisation
WO2011072099A2 (fr) 2009-12-09 2011-06-16 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase
WO2011094690A1 (fr) 2010-01-29 2011-08-04 The Procter & Gamble Company Film amélioré soluble dans l'eau comprenant un mélange de polymères pvoh, et paquets constitués de celui-ci
WO2011127102A1 (fr) 2010-04-06 2011-10-13 The Procter & Gamble Company Libération optimisée de systèmes de blanchiment dans des détergents de blanchisserie
WO2011140364A1 (fr) 2010-05-06 2011-11-10 Danisco Us Inc. Compositions et procédés comprenant des variants de la subtilisine
WO2011163428A1 (fr) 2010-06-24 2011-12-29 The Procter & Gamble Company Articles de type doses unitaires solubles comprenant un polymère cationique
WO2012027404A1 (fr) 2010-08-23 2012-03-01 The Sun Products Corporation Compositions de détergent en doses unitaires et leurs procédés de production et d'utilisation
WO2012059336A1 (fr) 2010-11-03 2012-05-10 Henkel Ag & Co. Kgaa Article pour le linge ayant des propriétés de nettoyage
WO2012104613A1 (fr) 2011-01-31 2012-08-09 Reckitt Benckiser N.V. Article de nettoyage
WO2012151534A1 (fr) 2011-05-05 2012-11-08 Danisco Us Inc. Procédés et compositions comprenant des variants de la sérine protéase
WO2013063460A2 (fr) 2011-10-28 2013-05-02 Danisco Us Inc. Variants d'alpha-amylase pour obtention de maltohexaose variant
US8530219B2 (en) 2008-11-11 2013-09-10 Danisco Us Inc. Compositions and methods comprising a subtilisin variant
WO2013184577A1 (fr) 2012-06-08 2013-12-12 Danisco Us Inc. Variants d'alpha-amylase dérivés de l'alpha-amylase de cytophaga sp. amylase/ (cspamy2)
WO2014071410A1 (fr) 2012-11-05 2014-05-08 Danisco Us Inc. Compositions et procédés comportant des variants de thermolysine protéase
WO2014099523A1 (fr) 2012-12-21 2014-06-26 Danisco Us Inc. Variants d'alpha-amylase
WO2014164777A1 (fr) 2013-03-11 2014-10-09 Danisco Us Inc. Variantes combinatoires d'alpha-amylases
WO2014194054A1 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194032A1 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194117A2 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194034A2 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2015038792A1 (fr) 2013-09-12 2015-03-19 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase lg12-clade
WO2015077126A1 (fr) 2013-11-20 2015-05-28 Danisco Us Inc. Variants d'alpha-amylases ayant une sensibilité réduite au clivage protéasique, et leurs procédés d'utilisation
WO2015089441A1 (fr) 2013-12-13 2015-06-18 Danisco Us Inc. Sérine protéases d'espèce de bacillus
WO2015089447A1 (fr) 2013-12-13 2015-06-18 Danisco Us Inc. Sérines protéases du clade du bacillus gibsonii
WO2015143360A2 (fr) 2014-03-21 2015-09-24 Danisco Us Inc. Sérine-protéases de l'espèce bacillus
WO2016001449A1 (fr) 2014-07-04 2016-01-07 Novozymes A/S Variants de subtilase et polynucléotides codant pour ceux-ci
WO2016001450A2 (fr) 2014-07-04 2016-01-07 Novozymes A/S Variants de subtilase et polynucléotides codant pour ceux-ci
WO2016007929A2 (fr) 2014-07-11 2016-01-14 Danisco Us Inc. Mannanases de paenibacillus et bacillus spp.
WO2016061438A1 (fr) 2014-10-17 2016-04-21 Danisco Us Inc. Sérines protéases de l'espèce bacillus
WO2016069548A2 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérine-protéases
WO2016069557A1 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérine-protéases de l'espèce bacillus
WO2016069569A2 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérine protéases
WO2016069544A1 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérines protéases
WO2016069552A1 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérines protéases
WO2016069563A1 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérine protéases
WO2016087617A1 (fr) 2014-12-04 2016-06-09 Novozymes A/S Variants de subtilase et polynucléotides codant pour ceux-ci
WO2016087619A1 (fr) 2014-12-04 2016-06-09 Novozymes A/S Compositions de nettoyage liquides comprenant des variants de protéase
WO2016145428A1 (fr) 2015-03-12 2016-09-15 Danisco Us Inc Compositions et procédés comprenant des variants de protéase lg12-clade
WO2016174234A2 (fr) 2015-04-29 2016-11-03 Novozymes A/S Polypeptides appropriés pour détergent
WO2016183509A1 (fr) 2015-05-13 2016-11-17 Danisco Us Inc. Variants de protéase aprl-clade et leurs utilisations
WO2016202835A2 (fr) 2015-06-18 2016-12-22 Henkel Ag & Co. Kgaa Composition de détergent comprenant des variants de subtilase
WO2016205755A1 (fr) 2015-06-17 2016-12-22 Danisco Us Inc. Protéases à sérines du clade du bacillus gibsonii
WO2017114891A1 (fr) * 2015-12-30 2017-07-06 Novozymes A/S Variants d'enzymes et polynucléotides codant pour ces variants
WO2018184004A1 (fr) * 2017-03-31 2018-10-04 Danisco Us Inc Variants combinatoires d'alpha-amylases

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02222687A (ja) * 1989-02-25 1990-09-05 Lotte Co Ltd 欠失型α―アミラーゼ
CN101010688A (zh) 2004-06-29 2007-08-01 科汀科技大学 定价新奇期权的方法和系统
EP2726500B1 (fr) * 2011-06-30 2019-03-20 Novozymes A/S Procédé de criblage d'alpha-amylases
WO2015044448A1 (fr) 2013-09-30 2015-04-02 Novozymes A/S Variants d'alpha-amylase et polynucléotides les codant
DK3387124T3 (da) * 2015-12-09 2021-08-23 William Cuevas Kombinatoriske alfa-amylasevarianter
EP3668973A2 (fr) * 2017-08-18 2020-06-24 Danisco US Inc. Variants d'alpha-amylases
US12410385B2 (en) * 2019-10-24 2025-09-09 The Procter & Gamble Company Automatic dishwashing detergent composition comprising an amylase
WO2023114792A1 (fr) * 2021-12-16 2023-06-22 The Procter & Gamble Company Composition pour d'entretien ménager comprenant une amylase

Patent Citations (164)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1372034A (en) 1970-12-31 1974-10-30 Unilever Ltd Detergent compositions
US4435307A (en) 1980-04-30 1984-03-06 Novo Industri A/S Detergent cellulase
US5700676A (en) 1984-05-29 1997-12-23 Genencor International Inc. Modified subtilisins having amino acid alterations
USRE34606E (en) 1984-05-29 1994-05-10 Genencor, Inc. Modified enzymes and methods for making same
US5955340A (en) 1984-05-29 1999-09-21 Genencor International, Inc. Modified subtilisins having amino acid alterations
EP0218272A1 (fr) 1985-08-09 1987-04-15 Gist-Brocades N.V. Enzymes lipolytiques et leur usage dans des compositions détergentes
EP0258068A2 (fr) 1986-08-29 1988-03-02 Novo Nordisk A/S Additif enzymatique pour détergent
EP0260105A2 (fr) 1986-09-09 1988-03-16 Genencor, Inc. Préparation d'enzymes à activité modifiée
EP0305216A1 (fr) 1987-08-28 1989-03-01 Novo Nordisk A/S Lipase recombinante de humicola et procédé de production de lipases recombinantes de humicola
WO1989006279A1 (fr) 1988-01-07 1989-07-13 Novo-Nordisk A/S Genes de subtilisine mutes
EP0331376A2 (fr) 1988-02-28 1989-09-06 Amano Pharmaceutical Co., Ltd. ADN recombinant, bactérie du genre pseudomonas le contenant et son utilisation dans un procédé de production de lipase
US5691178A (en) 1988-03-22 1997-11-25 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase
US5776757A (en) 1988-03-24 1998-07-07 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase and method of making thereof
US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
WO1989009259A1 (fr) 1988-03-24 1989-10-05 Novo-Nordisk A/S Preparation de cellulase
EP0407225A1 (fr) 1989-07-07 1991-01-09 Unilever Plc Enzymes et compositions détergentes enzymatiques
WO1991016422A1 (fr) 1990-04-14 1991-10-31 Kali-Chemie Aktiengesellschaft Lipases bacillaires alcalines, sequences d'adn de codage pour celles-ci et bacilles produisant ces lipases
US5457046A (en) 1990-05-09 1995-10-10 Novo Nordisk A/S Enzyme capable of degrading cellullose or hemicellulose
US5763254A (en) 1990-05-09 1998-06-09 Novo Nordisk A/S Enzyme capable of degrading cellulose or hemicellulose
EP0531315A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Enzyme capable de degrader la cellulose ou l"hemicellulose.
US5686593A (en) 1990-05-09 1997-11-11 Novo Nordisk A/S Enzyme capable of degrading cellulose or hemicellulose
EP0531372A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Preparation de cellulase comprenant un enzyme d'endoglucanase.
WO1992005249A1 (fr) 1990-09-13 1992-04-02 Novo Nordisk A/S Variantes lipasiques
EP0495257A1 (fr) 1991-01-16 1992-07-22 The Procter & Gamble Company Compositions de détergent compactes contenant de la cellulase de haute activité
WO1992021760A1 (fr) 1991-05-29 1992-12-10 Cognis, Inc. Enzymes proteolytiques mutantes tirees de bacillus
US5340735A (en) 1991-05-29 1994-08-23 Cognis, Inc. Bacillus lentus alkaline protease variants with increased stability
US5500364A (en) 1991-05-29 1996-03-19 Cognis, Inc. Bacillus lentus alkaline protease varints with enhanced stability
WO1993024618A1 (fr) 1992-06-01 1993-12-09 Novo Nordisk A/S Variante de peroxydase avec stabilite amelioree vis-a-vis du peroxyde d'hydrogene
WO1994001541A1 (fr) 1992-07-06 1994-01-20 Novo Nordisk A/S Lipase de c. antarctica et variantes lipasiques
WO1994007998A1 (fr) 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase
WO1994018314A1 (fr) 1993-02-11 1994-08-18 Genencor International, Inc. Alpha-amylase stable a l'oxydation
WO1994025578A1 (fr) 1993-04-27 1994-11-10 Gist-Brocades N.V. Nouveaux variants de lipase utilises dans des detergents
WO1995006720A1 (fr) 1993-08-30 1995-03-09 Showa Denko K.K. Nouvelle lipase, micro-organisme la produisant, procede de production de cette lipase, et utilisation de ladite lipase
WO1995010603A1 (fr) 1993-10-08 1995-04-20 Novo Nordisk A/S Variants d'amylase
WO1995010602A1 (fr) 1993-10-13 1995-04-20 Novo Nordisk A/S Variants de peroxydase stables par rapport a h2o¿2?
WO1995014783A1 (fr) 1993-11-24 1995-06-01 Showa Denko K.K. Gene de lipase et lipase variante
WO1995022615A1 (fr) 1994-02-22 1995-08-24 Novo Nordisk A/S Procede pour preparer un variant d'une enzyme lipolytique
WO1995023221A1 (fr) 1994-02-24 1995-08-31 Cognis, Inc. Enzymes ameliorees et detergents les contenant
US5801039A (en) 1994-02-24 1998-09-01 Cognis Gesellschaft Fuer Bio Und Umwelttechnologie Mbh Enzymes for detergents
WO1995024471A1 (fr) 1994-03-08 1995-09-14 Novo Nordisk A/S Nouvelles cellulases alcalines
WO1995026397A1 (fr) 1994-03-29 1995-10-05 Novo Nordisk A/S Amylase alcaline issue d'un bacille
WO1995030744A2 (fr) 1994-05-04 1995-11-16 Genencor International Inc. Lipases a resistance aux tensioactifs amelioree
US6602842B2 (en) 1994-06-17 2003-08-05 Genencor International, Inc. Cleaning compositions containing plant cell wall degrading enzymes and their use in cleaning methods
WO1995035381A1 (fr) 1994-06-20 1995-12-28 Unilever N.V. Lipases modifiees provenant de pseudomonas et leur utilisation
WO1996000292A1 (fr) 1994-06-23 1996-01-04 Unilever N.V. Pseudomonas lipases modifiees et leur utilisation
WO1996011262A1 (fr) 1994-10-06 1996-04-18 Novo Nordisk A/S Enzyme et preparation enzymatique presentant une activite endoglucanase
WO1996012012A1 (fr) 1994-10-14 1996-04-25 Solvay S.A. Lipase, micro-organisme la produisant, procede de preparation de cette lipase et utilisation de celle-ci
WO1996013580A1 (fr) 1994-10-26 1996-05-09 Novo Nordisk A/S Enzyme a activite lipolytique
US5855625A (en) 1995-01-17 1999-01-05 Henkel Kommanditgesellschaft Auf Aktien Detergent compositions
WO1996023873A1 (fr) 1995-02-03 1996-08-08 Novo Nordisk A/S Alleles d'amylase-alpha
WO1996023874A1 (fr) 1995-02-03 1996-08-08 Novo Nordisk A/S Technique de mise au point de mutants d'amylase-alpha dotes de proprietes predefinies
WO1996027002A1 (fr) 1995-02-27 1996-09-06 Novo Nordisk A/S Nouveau gene de lipase et procede de production de lipase a l'aide de celui-ci
WO1996029397A1 (fr) 1995-03-17 1996-09-26 Novo Nordisk A/S Nouvelles endoglucanases
WO1997004079A1 (fr) 1995-07-14 1997-02-06 Novo Nordisk A/S Enzyme modifiee a activite lipolytique
US6077316A (en) 1995-07-19 2000-06-20 Novo Nordisk A/S Treatment of fabrics
WO1997007202A1 (fr) 1995-08-11 1997-02-27 Novo Nordisk A/S Nouvelles enzymes lipolytiques
WO1997041213A1 (fr) 1996-04-30 1997-11-06 Novo Nordisk A/S MUTANTS DUNE AMYLASE-$g(a)
WO1998008940A1 (fr) 1996-08-26 1998-03-05 Novo Nordisk A/S Nouvelle endoglucanase
WO1998012307A1 (fr) 1996-09-17 1998-03-26 Novo Nordisk A/S Variants de cellulase
WO1998013481A1 (fr) 1996-09-26 1998-04-02 Novo Nordisk A/S Enzyme a activite amylase
WO1998015257A1 (fr) 1996-10-08 1998-04-16 Novo Nordisk A/S Derives de l'acide diaminobenzoique en tant que precurseurs de matieres tinctoriales
WO1999019467A1 (fr) 1997-10-13 1999-04-22 Novo Nordisk A/S MUTANTS D'α-AMYLASE
US6312936B1 (en) 1997-10-23 2001-11-06 Genencor International, Inc. Multiply-substituted protease variants
US6482628B1 (en) 1997-10-23 2002-11-19 Genencor International, Inc. Multiply-substituted protease variants
WO1999023211A1 (fr) 1997-10-30 1999-05-14 Novo Nordisk A/S Mutants d'alpha-amylase
WO1999042567A1 (fr) 1998-02-18 1999-08-26 Novo Nordisk A/S Amylase bacillaire alcaline
WO1999046399A1 (fr) 1998-03-09 1999-09-16 Novo Nordisk A/S Preparation enzymatique de sirop de glucose a partir d'amidon
US6566114B1 (en) 1998-06-10 2003-05-20 Novozymes, A/S Mannanases
US6730646B1 (en) 1998-07-29 2004-05-04 Reckitt Benckiser N.V. Composition for use in a dishwasher
WO2000029560A1 (fr) 1998-11-16 2000-05-25 Novozymes A/S VARIANTES DE α-AMYLASE
WO2000060059A2 (fr) 1999-03-30 2000-10-12 NovozymesA/S Variantes d'alpha amylase
WO2000060058A2 (fr) 1999-03-31 2000-10-12 Novozymes A/S Polypeptides possedant une activite alcaline alpha-amylase et acides nucleiques codant pour ces polypeptides
WO2000060060A2 (fr) 1999-03-31 2000-10-12 Novozymes A/S Polypeptides presentant une activite alcaline alpha-amylase et acides nucleiques les codant
WO2001014629A1 (fr) 1999-08-20 2001-03-01 Genencor International, Inc. Modification enzymatique de la surface d'une fibre ou d'un article de polyester
WO2001014532A2 (fr) 1999-08-20 2001-03-01 Novozymes A/S Amylase alcaline de bacillus
WO2001034899A1 (fr) 1999-11-05 2001-05-17 Genencor International, Inc. Enzymes utiles pour changer les proprietes du polyester
US6933140B1 (en) 1999-11-05 2005-08-23 Genencor International, Inc. Enzymes useful for changing the properties of polyester
WO2001064852A1 (fr) * 2000-03-03 2001-09-07 Novozymes A/S Polypeptides possedant une activite de l'alpha-amylase et acides nucleiques codant pour ces polypeptides
WO2001066712A2 (fr) 2000-03-08 2001-09-13 Novozymes A/S Variants possedant des proprietes modifiees
WO2001085888A2 (fr) 2000-05-11 2001-11-15 The Procter & Gamble Company Systeme de lessive a dosage unitise
WO2001088107A2 (fr) 2000-05-12 2001-11-22 Novozymes A/S Variantes d'alpha-amylase avec une activite 1,6 alteree
WO2001096537A2 (fr) 2000-06-14 2001-12-20 Novozymes A/S Alpha-amylase pre-oxydee
WO2002010355A2 (fr) 2000-08-01 2002-02-07 Novozymes A/S Mutants d'alpha-amylase a proprietes modifiees
US6440991B1 (en) 2000-10-02 2002-08-27 Wyeth Ethers of 7-desmethlrapamycin
EP1504994A2 (fr) 2000-11-27 2005-02-09 The Procter & Gamble Company Procédé de fabrication d'un sachet soluble dans l'eau
US20100081598A1 (en) 2000-11-27 2010-04-01 The Procter & Gamble Company Dishwashing method
WO2002092797A2 (fr) 2001-05-15 2002-11-21 Novozymes A/S Variant d'alpha-amylases ayant des proprietes modifiees
WO2003089562A1 (fr) 2002-04-19 2003-10-30 The Procter & Gamble Company Sachets de produit de nettoyage
WO2004055178A1 (fr) 2002-12-17 2004-07-01 Novozymes A/S Alpha-amylases thermostables
US7833773B2 (en) 2003-04-30 2010-11-16 Danisco Us Inc. Bacillus mHKcel cellulase
US7449318B2 (en) 2003-04-30 2008-11-11 Danisco A/S, Genencor Division Bacillus mHKcel cellulase
US7001878B2 (en) 2003-09-22 2006-02-21 The Procter & Gamble Company Liquid unit dose detergent composition
WO2005054475A1 (fr) 2003-12-03 2005-06-16 Meiji Seika Kaisha, Ltd. Stce d'endoglucanase et preparation de cellulase le contenant
WO2005056787A1 (fr) 2003-12-08 2005-06-23 Meiji Seika Kaisha, Ltd. Cellulase supportant les tensioactifs et procede de transformation associe
EP1740690A1 (fr) 2004-04-28 2007-01-10 Henkel Kommanditgesellschaft auf Aktien Procede pour produire des produits detergents ou nettoyants
WO2006002643A2 (fr) 2004-07-05 2006-01-12 Novozymes A/S Variants d'alpha-amylases presentant des proprietes modifiees
WO2006007911A1 (fr) 2004-07-20 2006-01-26 Unilever Plc Produit de blanchisserie
WO2006045391A1 (fr) 2004-10-29 2006-05-04 Unilever Plc Procede de preparation d'un produit de lessive
WO2007044993A2 (fr) 2005-10-12 2007-04-19 Genencor International, Inc. Utilisation et production d'une metalloprotease neutre stable au stockage
US20090209445A1 (en) 2006-03-22 2009-08-20 The Procter & Gamble Company Liquid treatment unitized dose composition
WO2008000567A1 (fr) 2006-06-30 2008-01-03 Unilever Plc Articles de lessive
US20080090747A1 (en) 2006-07-18 2008-04-17 Pieter Augustinus Protease variants active over a broad temperature range
WO2008010925A2 (fr) 2006-07-18 2008-01-24 Danisco Us, Inc., Genencor Division Variantes de protéases actives sur une large plage de températures
WO2008087426A1 (fr) 2007-01-18 2008-07-24 Reckitt Benckiser N.V. Élément de dosage et son procédé de fabrication
US20080293607A1 (en) 2007-03-09 2008-11-27 Jones Brian E Alkaliphilic Bacillus Species alpha-Amylase Variants, Compositions Comprising alpha-Amylase Variants, And Methods of Use
WO2009058661A1 (fr) 2007-10-31 2009-05-07 Danisco Us Inc., Genencor Division Utilisation et production de métalloprotéases neutres stables vis-à-vis des citrates
WO2009058303A2 (fr) 2007-11-01 2009-05-07 Danisco Us Inc., Genencor Division Production de thermolysine et de ses variants et utilisation dans des détergents liquides
WO2009061380A2 (fr) 2007-11-05 2009-05-14 Danisco Us Inc., Genencor Division Variants de bacillus sp. ts-23 alpha-amylase à propriétés modifiées
WO2009098659A1 (fr) 2008-02-08 2009-08-13 The Procter & Gamble Company Procédé de fabrication d'une poche soluble dans l'eau
WO2009098660A1 (fr) 2008-02-08 2009-08-13 The Procter & Gamble Company Sachet soluble dans l'eau
WO2009112992A1 (fr) 2008-03-14 2009-09-17 The Procter & Gamble Company Composition détergente pour lave-vaisselle
WO2009124160A1 (fr) 2008-04-02 2009-10-08 The Procter & Gamble Company Poche hydrosoluble renfermant une composition détergente
WO2009152031A1 (fr) 2008-06-13 2009-12-17 The Procter & Gamble Company Poche multi-compartiments
US8530219B2 (en) 2008-11-11 2013-09-10 Danisco Us Inc. Compositions and methods comprising a subtilisin variant
WO2010059483A1 (fr) 2008-11-20 2010-05-27 The Procter & Gamble Company Produits nettoyants
WO2010088112A1 (fr) 2009-01-28 2010-08-05 The Procter & Gamble Company Composition de poche à plusieurs compartiments pour lessive
WO2010090915A1 (fr) 2009-02-09 2010-08-12 The Procter & Gamble Company Composition détergente
WO2010115028A2 (fr) 2009-04-01 2010-10-07 Danisco Us Inc. Système de lavage comprenant une alpha-amylase et une protéase
WO2010116139A1 (fr) 2009-04-09 2010-10-14 Reckitt Benckiser N.V. Composition détergente
WO2010135238A1 (fr) 2009-05-19 2010-11-25 The Procter & Gamble Company Procédé d'impression d'un film soluble dans l'eau
WO2011013022A1 (fr) 2009-07-28 2011-02-03 Koninklijke Philips Electronics N.V. Unité de lavage et de stérilisation
WO2011072099A2 (fr) 2009-12-09 2011-06-16 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase
WO2011094690A1 (fr) 2010-01-29 2011-08-04 The Procter & Gamble Company Film amélioré soluble dans l'eau comprenant un mélange de polymères pvoh, et paquets constitués de celui-ci
WO2011094687A1 (fr) 2010-01-29 2011-08-04 The Procter & Gamble Company Film hydrosoluble ayant des propriétés de dissolution et de contrainte améliorées, et paquets fabriqués à partir de celui-ci
WO2011127102A1 (fr) 2010-04-06 2011-10-13 The Procter & Gamble Company Libération optimisée de systèmes de blanchiment dans des détergents de blanchisserie
WO2011140364A1 (fr) 2010-05-06 2011-11-10 Danisco Us Inc. Compositions et procédés comprenant des variants de la subtilisine
WO2011163428A1 (fr) 2010-06-24 2011-12-29 The Procter & Gamble Company Articles de type doses unitaires solubles comprenant un polymère cationique
WO2012027404A1 (fr) 2010-08-23 2012-03-01 The Sun Products Corporation Compositions de détergent en doses unitaires et leurs procédés de production et d'utilisation
WO2012059336A1 (fr) 2010-11-03 2012-05-10 Henkel Ag & Co. Kgaa Article pour le linge ayant des propriétés de nettoyage
WO2012104613A1 (fr) 2011-01-31 2012-08-09 Reckitt Benckiser N.V. Article de nettoyage
WO2012151534A1 (fr) 2011-05-05 2012-11-08 Danisco Us Inc. Procédés et compositions comprenant des variants de la sérine protéase
WO2013063460A2 (fr) 2011-10-28 2013-05-02 Danisco Us Inc. Variants d'alpha-amylase pour obtention de maltohexaose variant
WO2013184577A1 (fr) 2012-06-08 2013-12-12 Danisco Us Inc. Variants d'alpha-amylase dérivés de l'alpha-amylase de cytophaga sp. amylase/ (cspamy2)
WO2014071410A1 (fr) 2012-11-05 2014-05-08 Danisco Us Inc. Compositions et procédés comportant des variants de thermolysine protéase
WO2014099523A1 (fr) 2012-12-21 2014-06-26 Danisco Us Inc. Variants d'alpha-amylase
WO2014164777A1 (fr) 2013-03-11 2014-10-09 Danisco Us Inc. Variantes combinatoires d'alpha-amylases
WO2014194054A1 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194032A1 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194117A2 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194034A2 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2015038792A1 (fr) 2013-09-12 2015-03-19 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase lg12-clade
WO2015077126A1 (fr) 2013-11-20 2015-05-28 Danisco Us Inc. Variants d'alpha-amylases ayant une sensibilité réduite au clivage protéasique, et leurs procédés d'utilisation
WO2015089441A1 (fr) 2013-12-13 2015-06-18 Danisco Us Inc. Sérine protéases d'espèce de bacillus
WO2015089447A1 (fr) 2013-12-13 2015-06-18 Danisco Us Inc. Sérines protéases du clade du bacillus gibsonii
WO2015143360A2 (fr) 2014-03-21 2015-09-24 Danisco Us Inc. Sérine-protéases de l'espèce bacillus
WO2016001449A1 (fr) 2014-07-04 2016-01-07 Novozymes A/S Variants de subtilase et polynucléotides codant pour ceux-ci
WO2016001450A2 (fr) 2014-07-04 2016-01-07 Novozymes A/S Variants de subtilase et polynucléotides codant pour ceux-ci
WO2016007929A2 (fr) 2014-07-11 2016-01-14 Danisco Us Inc. Mannanases de paenibacillus et bacillus spp.
WO2016061438A1 (fr) 2014-10-17 2016-04-21 Danisco Us Inc. Sérines protéases de l'espèce bacillus
WO2016069569A2 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérine protéases
WO2016069557A1 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérine-protéases de l'espèce bacillus
WO2016069548A2 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérine-protéases
WO2016069544A1 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérines protéases
WO2016069552A1 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérines protéases
WO2016069563A1 (fr) 2014-10-27 2016-05-06 Danisco Us Inc. Sérine protéases
WO2016087619A1 (fr) 2014-12-04 2016-06-09 Novozymes A/S Compositions de nettoyage liquides comprenant des variants de protéase
WO2016087617A1 (fr) 2014-12-04 2016-06-09 Novozymes A/S Variants de subtilase et polynucléotides codant pour ceux-ci
WO2016145428A1 (fr) 2015-03-12 2016-09-15 Danisco Us Inc Compositions et procédés comprenant des variants de protéase lg12-clade
WO2016174234A2 (fr) 2015-04-29 2016-11-03 Novozymes A/S Polypeptides appropriés pour détergent
WO2016183509A1 (fr) 2015-05-13 2016-11-17 Danisco Us Inc. Variants de protéase aprl-clade et leurs utilisations
WO2016205755A1 (fr) 2015-06-17 2016-12-22 Danisco Us Inc. Protéases à sérines du clade du bacillus gibsonii
WO2016202835A2 (fr) 2015-06-18 2016-12-22 Henkel Ag & Co. Kgaa Composition de détergent comprenant des variants de subtilase
WO2017114891A1 (fr) * 2015-12-30 2017-07-06 Novozymes A/S Variants d'enzymes et polynucléotides codant pour ces variants
WO2018184004A1 (fr) * 2017-03-31 2018-10-04 Danisco Us Inc Variants combinatoires d'alpha-amylases

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
DARTOIS ET AL., BIOCHEMICA ETBIOPHYSICA ACTA, vol. 1131, 1993, pages 253 - 360
EDGAR, R.C., BIOINFORMATICS, vol. 5, 2004, pages 113
EDGAR, R.C., NUC. ACIDS RES., vol. 32, 2004, pages 1792 - 97
HO, M-C. ET AL.: "The origin of the electrostatic perturbation in acetoacetate decarboxylase", NATURE, vol. 459, 2009, pages 393 - 97
JENCKS, W.P.: "Catalysis in Chemistry and Enzymology", 1986, DOVER PUBLICATIONS
NIELSEN J E ET AL: "Protein engineering of bacterial @a-amylases", BIOCHIMICA ET BIOPHYSICA ACTA. PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, ELSEVIER, AMSTERDAM; NL, vol. 1543, no. 2, 29 December 2000 (2000-12-29), pages 253 - 274, XP004279109, ISSN: 0167-4838, DOI: 10.1016/S0167-4838(00)00240-5 *
NIELSEN, J.E.BORCHERT, T.V.: "Protein engineering of bacterial a-amylases", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1543, 2000, pages 253 - 274, XP004279109, doi:10.1016/S0167-4838(00)00240-5
RYDBERG, E.H. ET AL.: "Mechanistic analyses of catalysis in human pancreatic a-amylases: Detailed kinetic and structural studies of three conserved carboxylic acids", BIOCHEMISTRY, vol. 41, 2002, pages 4492 - 4502
SCHMIDT, D.E.WESTHEIMER, F.H.: "pK of the lysine amino group at the active site of acetoacetate decarboxylase", BIOCHEMISTRY, vol. 10, 1971, pages 1249 - 53
SUZUKI ET AL., J. BIOL. CHEM., vol. 264, 1989, pages 18933 - 938
THOMPSON ET AL., NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 4680

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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